Co‐localization of clinically relevant antibiotic‐ and heavy metal resistance genes on plasmids in Klebsiella pneumoniae from marine bivalves

Abstract Klebsiella pneumoniae is an opportunistic pathogen frequently associated with antibiotic resistance and present in a wide range of environments, including marine habitats. However, little is known about the development, persistence, and spread of antibiotic resistance in such environments. This study aimed to obtain the complete genome sequences of antibiotic‐resistant K. pneumoniae isolated from marine bivalves in order to determine the genetic context of antibiotic‐ and heavy metal resistance genes in these isolates. Five antibiotic‐resistant K. pneumoniae isolates, of which four also carried heavy metal resistance genes, were selected for complete genome sequencing using the Illumina MiSeq platform and the Oxford Nanopore Technologies GridION device. Conjugation experiments were conducted to examine the transfer potential of selected plasmids. The average length of the complete genomes was 5.48 Mbp with a mean chromosome size of 5.27 Mbp. Seven plasmids were detected in the antibiotic‐resistant isolates. Three IncFIB, one IncFIB/IncFII, and one IncFIB/IncHIB plasmid, respectively, carried antibiotic resistance genes such as qnrS1, aph(6)‐Id and aph(3′)‐Ia, aadA1, and aadA2. Four of these plasmids also carried genes encoding resistance to copper (pco), silver (sil), and arsenic (ars). One plasmid carrying tet(D) and bla SHV‐1 as well as pco, sil, and ars genes was transferred to Escherichia coli by conjugation. We show the co‐occurrence of antibiotic‐ and heavy metal resistance genes on a conjugative IncFIB plasmid from K. pneumoniae from marine bivalves. Our study highlights the importance of the marine environment and seafood as a possible dissemination route for antimicrobial resistance and provides insights into the potential for co‐selection of antibiotic resistance genes by heavy metals.


| INTRODUCTION
Klebsiella pneumoniae is an opportunistic pathogen and a common cause of nosocomial infections. K. pneumoniae is often associated with antibiotic resistance, and strains resistant to clinically important antibiotics are considered a critical threat to public health (Wyres et al., 2020). K. pneumoniae is commonly found in the gastrointestinal tract of humans and animals but can also be isolated from a range of environments, including soil, plants, surface waters, and marine organisms (Brisse et al., 2006;Håkonsholm et al., 2022;Wyres et al., 2020).
Increased resistance to antibiotics is one of the greatest threats in modern medicine (Church & McKillip, 2021). Infections caused by antibiotic-resistant bacteria were estimated to be the direct cause of 1.27 million deaths globally in 2019 (Murray et al., 2022). K.
pneumoniae is considered an important contributor to the spread of antibiotic resistance (Wyres & Holt, 2018). Resistance to broadspectrum cephalosporins and carbapenems is increasingly reported in clinical K. pneumoniae isolates in the WHO European region, with 44% of countries reporting resistance rates of ≥50% to thirdgeneration cephalosporins in 2020, particularly in southern and eastern European countries. However, the occurrence is still low in Scandinavian countries with an average of 8.1% invasive K.
pneumoniae isolates resistant to third-generation cephalosporins (WHO Regional office for Europe, ECDC, 2021).
Horizontal gene transfer is one of the primary drivers of antibiotic resistance, and the spread of antibiotic-resistance genes (ARGs) is driven by conjugative plasmids (San Millan, 2018). Plasmids can be classified according to their incompatibility (Inc), which refers to their inability to co-exist stably in the same cell line over time. In general, closely related plasmids are often incompatible, while those more distantly related often are compatible. Overall, 28 Inc groups have been reported within the Enterobacterales family, and some of these are frequently associated with ARGs, for example, extendedspectrum β-lactamase (ESBL) and carbapenemase-encoding genes are commonly found on IncF plasmids (Carattoli, 2009;Rozwandowicz et al., 2018). In K. pneumoniae, most of the ARGs are present on large conjugative plasmids, and most acquired ARGs are carried on plasmids belonging to the IncFII, IncN, IncR, and/or IncX3 groups (Wyres & Holt, 2018;Wyres et al., 2020).
The environment is recognized as important habitat for the development and spread of antibiotic resistance (Bengtsson-Palme et al., 2017;Marathe et al., 2017). Although overuse of antibiotics is a major driver of antibiotic resistance, other compounds such as heavy metals and biocides can cause co-selection of antibiotic-resistant bacteria. Unlike antibiotics, metals in the environment are not degraded, and their presence could therefore represent a long-term selection pressure (Baker-Austin et al., 2006).
In a previous study, we have shown the presence of antibioticresistant K. pneumoniae carrying heavy metal resistance genes (HMRGs) in bivalve mollusks and seawater from the Norwegian marine environment (Håkonsholm et al., 2022). The present study aimed to obtain complete genome sequences of K. pneumoniae isolates carrying ARGs and HMRGs using a combination of long-and short-read whole-genome sequencing in order to determine the genetic context of ARGs and HMRGs in this setting. We show the co-occurrence of ARGs and HMRGs on a conjugative IncFIB plasmid in one of the isolates.

| Bacterial isolates
Five K. pneumoniae sensu stricto isolates with acquired ARGs recovered from marine bivalves were selected for complete genome sequencing (Håkonsholm et al., 2022). Four isolates were recovered from blue mussels (Mytilus edulis) and one from oysters (Crassostrea gigas). Antibiotic susceptibility testing of the isolates was done by disk diffusion as described previously (Håkonsholm et al., 2020).

| Whole-genome sequencing, hybrid de novo assembly, and bioinformatic analyses
The short-read sequencing was performed as described previously (Håkonsholm et al., 2022). For the long-read sequencing, DNA was extracted manually using the Beckman Coulter Life Science GenFind V3 with the protocol: "DNA extraction from Bacteria using GenFindV3" (Beckman Coulter). Library preparation was done with the SQK-LSK-109 kit (Oxford Nanopore Technologies), DNA libraries were loaded onto a MINion flow cell (R9.4.1), and sequencing was done using the Oxford Nanopore Technologies GridION device.
Circular plasmid maps were created using the Proksee server (https://proksee.ca) and alignments of plasmid regions carrying HMRGs were generated with Easyfig v2.2.5 (Sullivan et al., 2011) using a minimum sequence identity of 80%.   Figure A1). Five plasmids, identified in five separate isolates, carried acquired ARGs, while four of these also carried genes encoding resistance to heavy metals. Phenotypic antibiotic susceptibility data for the isolates included in this study are provided in Supporting Information: Table S4. All acquired ARGs and HMRGs were co-located on IncFIB plasmids (Table 1).

| Comparison of plasmid regions carrying HMRGs
The to plasmid CP065035 (Håkonsholm et al., 2020). In plasmid pKp1792_2, the sil, pco, ars, clpK, and hsp20 genes were flanked by an IS5 transposase, whereas plasmid pKp1198 carried the sil and pco operon and heat tolerance genes in a region containing several different transposases and ars and mer were clustered with ARGs in a separate region flanked by IS26.

| Conjugation assay
Plasmid pKp319 from K. pneumoniae isolate 2016-319 was transferred to E. coli CV601-GFP strain, yielding transconjugants with identical resistance patterns (AMP R , TET R ) at a transfer frequency of 5.1 × 10 −4 transconjugants per recipient cell. Antibiotic susceptibility patterns of the obtained transconjugants are provided in Supporting Information: Table S1. Even though we predicted plasmid pKp1200_1 to be conjugative based on the genotype, we were not able to verify the conjugative transfer of this plasmid to the E. coli recipient in repeated experiments, indicating either a very low transfer frequency or inability of pKp1200_1 to transfer to E. coli.

| DISCUSSION
In the present study, we report the complete genome sequences of antibiotic-resistant K. pneumoniae isolated from bivalve mollusks collected along the Norwegian coast. We show the co-localization of ARGs and HMRGs on IncF plasmids, suggesting the potential for coselection of ARGs by heavy metals in the marine environment.
Five of the identified plasmids carried genes encoding resistance to aminoglycosides, sulfonamides, cephalosporins, tetracycline, quinolones, and/or amphenicols, all considered to be important by the WHO for the treatment of infections in humans (World Health Organization, 2019). Most of the plasmids carrying ARGs belonged to the IncF group. IncF is the most frequently described plasmid type, and is commonly found in bacteria of both human and animal origin (Rozwandowicz et al., 2018). Previous studies have also shown that IncF plasmid replicons are common in Norwegian K. pneumoniae isolates from clinics, wastewater, and from community-based carriers (Fostervold et al., 2021;Radisic et al., 2023;Raffelsberger et al., 2021).
Furthermore, a study on antibiotic-resistant E. coli in marine sediments and clams collected in Italy found IncF type plasmids as the most common plasmid type carrying ARGs, while another study found IncF plasmids among CTX-M producing E. coli and K.
Plasmids belonging to this Inc group are often associated with ARGs and are recognized as important contributors to the spread of antibiotic resistance, especially quinolone resistance genes, ESBLs, carbapenemases, and genes encoding resistance to aminoglycosides (Rozwandowicz et al., 2018). This is in accordance with our results, where three of the five resistance plasmids carried genes involved in resistance to quinolones, aminoglycosides, or β-lactam antibiotics, suggesting that such plasmids may be important in the dissemination of ARGs also in the marine environment.
Only plasmid pKp319 encoding resistance to tetracycline and ampicillin was transferred to the E. coli recipient via conjugation. The presence of ARGs and HMRGs on a conjugative plasmid indicates the potential for dissemination of such plasmids in the marine environment. However, we were not able to show the transmissibility of plasmid pKp1200_1, which carried multiple ARGs and genes related to conjugation to the recipient used in this study. This is in accordance with a previous study showing the inability of a CTX-M encoding IncFIB(K)/IncFII(K) plasmid (pKp848CTX), carrying a conserved transfer region, to transfer to an E. coli recipient in broth and filter mating experiments (Löhr et al., 2015). Further experiments using recipients belonging to different species/genera, including other Klebsiella spp., may be necessary to confirm the transferability of plasmid pKp1200_1. Additionally, pKp1200_1 carried genes encoding Klebicin B, a bacteriocin with nuclease activity, possibly reducing the number of recipient cells (Riley & Wertz, 2002;Riley et al., 2001).
Even though the plasmids reported in this study carry clinically relevant ARGs, one of the important findings of our study is the colocalization of ARGs and HMRGs on the same plasmids in K.
pneumoniae isolated from marine bivalves. HMRGs are frequently reported in clinical K. pneumoniae isolates as well as isolates from wastewater and marine environments, including seafood organisms (Furlan et al., 2020;Håkonsholm et al., 2022;Radisic et al., 2023;Sütterlin et al., 2017). Interestingly, most plasmids included in the present study carried similar regions harboring the sil and pco operon, and also genes encoding heat tolerance (hsp20, clpK), indicating that this region could be common in K. pneumoniae plasmids belonging to the IncFIB group. Furthermore, in plasmids pKp319, pKp1200_1, CP065035, and pKp848CTX the sil and pco operons were flanked by IS5 and truncated ISL3 transposases, possibly indicating that this composite transposon is important in the dissemination of heavy metal resistance, also in clinical settings (Löhr et al., 2015;Sütterlin et al., 2017). Overall, HMRGs were associated with different types of transposases in all plasmids, including members of the IS5 and IS26 families, indicating a potential for mobilization of these genes (Partridge et al., 2018).
Norway has a low prevalence of antibiotic resistance and low use of antibiotics in both human and veterinary medicine (NORM/ NORM-VET, 2020). However, heavy metals, such as copper, are used in the aquaculture industry, both in antifouling agents and as additives in fish feed (Grefsrud et al., 2021;Seiler & Berendonk, 2012). As a result, copper can contaminate the marine environment through fecal material, spilled feed, and leakage from metal-impregnated fish farm nets (Grefsrud et al., 2021). Copper is also naturally occurring in marine sediments and seawater (Grefsrud et al., 2021). Additionally, metal compounds, including arsenic, are used in wood preservation, livestock feed, and in agriculture as pesticides, fertilizers, and antimicrobials Seiler & Berendonk, 2012;Silbergeld & Nachman, 2008), and can therefore be spread to the marine environment through run-off from agricultural land. It has been suggested that pcoA and pcoB alone can confer copper resistance; however, pcoC, pcoD, and pcoE are required for full copper resistance (Argudín et al., 2019). The plasmids detected in our study harbored pcoABCDE, silABCEFPRS, and arsenic resistance genes. Previously, Gullberg et al. (2014) have shown that low concentrations of copper and especially arsenic were sufficient to maintain the multi-drug resistance pUUH239.2 plasmid (NC_016966), carrying ars and pco genes, from a K. pneumoniae strain responsible for a nosocomial outbreak in Sweden. Thus, our results indicate the potential for co-selection of ARGs in K.
pneumoniae in metal-contaminated marine environments.
Furthermore, all plasmids encoding both ARGs and HMRGs characterized in the present study carried type II TA systems, responsible for the killing, or growth-inhibition, of plasmid-free progeny cells (Kamruzzaman et al., 2021). These TA systems thus ensure that the plasmids are maintained and disseminated in bacterial populations even in environments without selection pressure imposed by antibiotics and/or heavy metals (Martinez, 2009). These plasmids can potentially persist in such environments and be transferred to human microbiota through, for example, seafood or direct contact via recreational activities.

| CONCLUSION
In the present study, we report the complete genome sequences of antibiotic-resistant K. pneumoniae isolated from marine bivalve mollusks collected along the Norwegian coast. We show colocalization of ARGs and HMRGs on IncFIB, IncFIB/IncFII, and IncFIB/IncHIB plasmids present in K. pneumoniae isolated from the marine bivalves. We further show that one of the plasmids carrying ARGs and HMRGs is transferrable to E. coli via conjugation. Our study shows the potential for co-selection of ARGs and/or antibioticresistant K. pneumoniae in the marine environment by heavy metals.
It also demonstrates the importance of the marine environment and seafood as dissemination routes for ARGs and pathogens and highlights the need for surveillance of antibiotic resistance in the marine environment.